1. Field of the Invention
The present invention relates to a construct comprising polyhydroxyalkanoate and a base material at least partly coated with the polyhydroxyalkanoate, and to a method for making the construct. More specifically, the present invention relates to a construct which comprises a base material that is at least partly coated with a polyhydroxyalkanoate made with a 3-hydroxyalkanoic acid monomer unit other than 3-hydroxypropionic acid unit, 3-hydroxy-n-butyric acid unit, or 3-hydroxy-n-valeric acid unit. The present invention also relates to a method for making the construct by immobilizing a polyhydroxyalkanoate synthetic enzyme that participates in medium chain length polyhydroxyalkanoate biosynthesis to the base material and carrying out synthesis of polyhydroxyalkanoate from 3-hydroxyacyl coenzyme A to coat at least part of the base material. Further, the construct of the present invention includes a capsule construct that comprises a particulate base material coated with polyhydroxyalkanoate, and a laminated construct that comprises a plate or film base material coated with polyhydroxyalkanoate.
Each construct of the present invention has a wide range of use as a functional construct. For example, the capsule construct can be used as a capsule toner for electrophotography, and the laminated construct can be used as a recording medium such as OHP film or inkjet recording medium.
The present invention also relates to electrostatic charge image developing toner employed in electrophotography, electrostatic recording or the like, a producing method for such toner and an image forming method utilizing such toner.
2. Related Background Art
Polymer materials are indispensable for today's life or industries. Polymer materials are utilized in various fields, for example, as housing materials for home electrics, packing materials, buffer materials and textile materials because of cheapness, light in weight and good formability. On the other hand, various polymer functional materials such as liquid crystals and coating materials have been produced utilizing the stable properties by introducing various functional substituents into polymer chains. These functional materials can expect larger market needs in a smaller production scale due to the higher added value than the bulk polymers as structural materials. Such polymeric functional materials have been produced by organic synthetic chemistry, for example, by introducing substituents during or after synthesis process of the polymer. Most polymers to be the skeleton of functional polymers are produced from petroleum-based raw materials by organic synthetic chemistry. Such polymers include polyethylene, polyethylene terephthalate, polyesters, polystyrene, polyvinylchloride, and polyacrylamide.
As one of the constituent techniques for confer higher added value to the polymer compound, the inventors of the present invention have been focusing on a layered construct in which a base material is coated with a polymer compound. By coating a certain base material a polymer compound, a composite construct having an extremely useful functionality can be obtained. Specific applications of such a construct include, for example, a capsule toner for electrophotography having a microcapsule structure encapsulating toner components in a polymer compound, and a recording medium for inkjet recording, where a sheet of base material is coated with a polymer compound.
Generally, in the electrophotography, an electrostatic latent image is formed by various means on a photosensitive material utilizing a photoconductive substance, then the latent image is developed with a toner, and the toner image is transferred to a transfer material such as paper according to the necessity and then fixed by heat, pressure, heat and pressure, or solvent vapor to obtain a copy image. As a toner used for this purpose, a “pulverized toner” has been used. The pulverized toner is produced by melting and mixing uniformly a colorant such as dyes and pigments in a thermoplastic resin, and then pulverizing the resin mixture by means of a pulverizer and a classifier to obtain a desired particle size. Although such a toner has excellent performance, there are some problems that, for example, the selection range of the materials is limited because brittleness is required the materials due to the making step in the toner production. In order to overcome such problems, Japanese Patent Publication No. 36-10231 or the like have suggested the manufacture of “a polymerized toner” by means of suspension polymerization. In the suspension polymerization, a polymerizable monomer, a colorant, a polymerization initiator, and if necessary a crosslinking agent and a charge controlling agent all of which are uniformly dissolved or dispersed are dispersed into a continuous phase (an aqueous phase, for example) containing a dispersion stabilizer by using an agitator, and polymerization reaction is carried out to obtain a desired toner. This method has advantages that, for example, brittleness is not required for the toner material because this method does not include pulverization step and consequently soft materials can be used. However, with such a polymerized toner of a fine particle size, the colorant and the charge controlling agent tend to be exposed on the toner surface, so that the toner tends to be affected by the colorant and reduction in uniform charging is concerned. In order to solve these problems, so-called “capsule toner” of which surface is coated with a polymer layer or layers has been proposed.
For example, Japanese Patent Application Laid-Open No. 8-286416, for example, discloses a capsule toner for electrostatic development and a production method in which polymerized particles are coated with a shell of a polar resin. In accordance with this method, a shell is formed around a core of a polymerized particle containing toner components by organic synthetic chemistry, by which the above described problem is overcome and an excellent capsule toner is obtained for electrostatic charge development with increased image durability and uniformity and stabilization of charging. In addition, Japanese Patent Application Laid-Open No. 9-292735 discloses a capsule toner for image formation in which a core made of a material of high thermal expansion and a release agent is covered with a hard resin layer. The above-described toner is a functional microcapsule designed such that the thermally expandable material within the core expands at fixation heating to break the shell and release the contained releasing agent out of the shell. Therefore, it can be expected that the above-described toner have such advantages that it can prevent offset when a film heating fixation apparatus is used, or it enables low-pressure fixation and alleviation of paper wrinkling when a roller fixing apparatus is used. Japanese Patent Application Laid-Open Nos. 5-119531, 5-249725, 6-332225, 9-43896, 10-78676, 11-7163, 2000-66444, 2000-112174, and 2000-330321 also disclose capsule constructs having a polymer shell and production methods thereof, all of which produce capsule toners by organic synthetic chemistry such as suspension polymerization, emulsion polymerization, precipitation polymerization, dispersion polymerization, soap-free emulsion polymerization, and seed polymerization.
However, these methods for making the capsule toners have some problems that the making process becomes extremely complicated and a large amount of solvents and surfactants are used in the making process.
On the other hand, the laminated construct comprised of a sheet of a base material coated with a polymer compound is used, for example, as a recording medium for inkjet recording system. In the inkjet recording system, micro-droplets of ink are applied to a recording medium such as paper to record images and characters on it by ejecting droplets on various operation principles. In such recording system, the ink contains a lot of liquid medium such as water and a mixture of water and organic solvents, so that the ink must be applied in a certain amount to obtain a high image density. In addition, since the ink droplets are continuously ejected to the recording medium, there may occur a beading phenomenon where the ejected ink droplets fuse and consequently dots will fuse leading to image distortion. Therefore, high ink-absorbing capacity and high ink-absorption speed are required for the inkjet recording medium.
For this purpose, it has been proposed a recording medium provided with an ink-receiving layer on the base material to increase ink absorption capacity. For example, Japanese Patent Application Laid-Open No. 55-146786 proposes a recording medium of which base material is coated with a water-soluble resin such as polyvinyl alcohol and polyvinyl pyrrolidone. In addition, Japanese Patent Application Laid-Open No. 5-221112 and others propose a recording medium employing a waterproof resin. Further, a recording medium employing an ionic resin as an ink receiving layer is proposed (Japanese Patent Application Laid-Open Nos. 11-78221 and 2000-190631, for example), to provide a recording medium excellent in wettability, water resistance, and dye-fixing properties, as well as the ink-absorption and drying properties and vivid image forming properties.
To form the ink absorbing layer on the base material, conventionally, coating methods have been widely used, for example, blade coating, air knife coating, roll coating, flash coating, gravure coating, kiss coating, die coating, extrusion coating, slide hopper coating, curtain coating, spray coating, etc.
In any of the methods mentioned above, the polymer compounds used for coating the base material are synthesized by organic synthesis, followed by addition of various functions.
Meanwhile, active studies have been done to produce polymer compounds by using biotechnology, and partly in practice. For example, known microbial polymers include polyhydroxyalkanoates (PHAs) such as poly-3-hydroxy-n-butyric acid (PHB), and copolymers of 3-hydroxy-n-butyric acid and 3-hydroxy-n-valeric acid (PHB/V); polysaccharides such as bacterial cellulose and pullulan; and polyamino acids such as poly-γ-glutamic acid and polylysine. Particularly, PHA can be processed into various products by melt-preparation etc., just like other existing plastics. In addition, because of excellent biocompatibility, application of PHA as medical soft materials is also expected.
It has been reported that many microorganisms produce PHA and accumulate it within cells. For example, microbial productions of PHB/V by Alcaligenes eutrophus H16 (ATCC No. 17699), Methylobacterium sp., Paracoccus sp., Alcaligenes sp., and Pseudomonas sp. have been reported (for example, Japanese Patent Application Laid-Open No. 5-74492, Japanese Patent Publication Nos. 6-15604, 7-14352, and 8-19227). Furthermore, Comamonas acidovorans IFO 13852 produces PHA comprised of monomer units of 3-hydroxy-n-butyric acid and 4-hydroxy-n-butyric acid (Japanese Patent Application Laid-Open No. 9-191893), and Aeromonas caviae produces a copolymer of 3-hydroxy-n-butyric acid and 3-hydroxyhexanoic acid (Japanese Patent Application Laid-Open Nos. 5-93049 and 7-265065).
Biosynthesis of these PHB and PHB/V is an enzymatic polymerization reaction using as a substrate (R)-3-hydroxybutyryl CoA or (R)-3-hydroxyvaleryl CoA that is synthesized from various carbon sources through various metabolic pathways within a living organism. The enzyme that catalyzes this polymerization reaction is PHB synthetase (this can be referred to as PHB polymerase or PHB synthase). CoA is an abbreviation for coenzyme A, and its chemical structure is represented by the following chemical formula.

Recently, active studies on polyhydroxyalkanoate comprised of 3-hydroxyalkanoic acid units of medium-chain-length (about 3 to 12 carbon atoms) (mcl-PHA) have been conducted.
For example, Japanese Patent No. 2642937 discloses that Pseudomonas oleovorans ATCC 29347 can produce PHA comprised of 3-hydroxyalkanoic acid monomer units of 6 to 12 carbon atoms from non-cyclic aliphatic hydrocarbons. In addition, it has been reported, in Appl. Environ. Microbiol., 58, 746 (1992), that Pseudomonas resinovorans produces PHA of which monomer units are 3-hydroxy-n-butyric acid, 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid, and 3-hydroxydecanoic acid using octanoic acid as a sole carbon source, and it also produces PHA of which monomer units are 3-hydroxy-n-butyric acid, 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid, and 3-hydroxydecanoic acid using hexanoic acid as sole carbon source. Here, the 3-hydroxyalkanoic acid monomer units longer than the raw material fatty acid are considered derived from the fatty acid synthesizing pathway described below.
Int. J. Biol. Macromol., 16 (3), 119 (1994) reported that Pseudomonas sp. Strain 61-3 produces PHA comprised of monomer units of 3-hydroxyalkanoic acids such as 3-hydroxy-n-butyric acid, 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid, and 3-hydroxydecanoic acid, and 3-hydroxyalkenoic acids such as 3-hydroxy-5-cis-decenoic acid and 3-hydroxy-5-cis-dodecenoic acid, using sodium gluconate as a sole carbon source.
The above-described PHAs are comprised of monomer units having alkyl groups as the side chain (usual-PHAs). However, when wider application of PHA, e.g., as a functional polymer, is intended, PHA having side chains other than alkyl groups (for example, side chains having substituents such as phenyl group, unsaturated hydrocarbons, ester groups, allyl group, cyano group, halogenated hydrocarbons, and epoxides) is extremely useful (unusual-PHA).
As for biosynthesis of unusual-PHA having phenyl groups, it was reported that Pseudomonas oleovorans produced PHA having 3-hydroxy-5-phenylvaleric acid units from 5-phenylvaleric acid (Polymers, 24, 5256–5260 (1991), Macromol. Chem., 191, 1957–1965 (1990); Chirality, 3, 492–494 (1991)). Polymers, 29, 1762–1766 (1996) reported that Pseudomonas oleovorans produced PHA having 3-hydroxy-5-(4-tolyl)valeric acid units from 5-(4-tolyl)valeric acid (5-(4-methylphenyl)valeric acid). Further, Polymers, 32, 2889–2895 (1999) reported that Pseudomonas oleovorans produced PHA having 3-hydroxy-5-(2,4-dinitrophenyl)valeric acid units and 3-hydroxy-5-(4-nitrophenyl)valeric acid units from 5-(2,4-dinitrophenyl)valeric acid.
As for unusual-PHA having phenoxy groups, Macromol. Chem. Phys., 195, 1665–1672 (1994) reported that Pseudomonas oleovorans produced PHA having 3-hydroxy-5-phenoxyvaleric acid units and 3-hydroxy-9-phenoxynonanoic acid units from 11-phenoxyundecanoic acid. Also, Polymers, 29, 3432–3435 (1996) reported that Pseudomonas oleovorans produced a PHA having 3-hydroxy-4-phenoxybutyric acid units and 3-hydroxy-6-phenoxyhexanoic acid units from 6-phenoxyhexanoic acid, a PHA having 3-hydroxy-4-phenoxybutyric acid units, 3-hydroxy-6-phenoxyhexanoic acid units, and 3-hydroxy-8-phenoxyoctanoic acid units from 8-phenoxyoctanoic acid, and a PHA having 3-hydroxy-5-phenoxyvaleric acid unit and 3-hydroxy-7-phenoxyheptanoic acid units from 11-phenoxyundecanoic acid. Further, Can. J. Microbiol., 41, 32–43 (1995) reported that Pseudomonas oleovorans ATCC 29347 and Pseudomonas putida KT 2442 produced PHA having 3-hydroxy-p-cyanophenoxyhexanoic acid units and PHA having 3-hydroxy-p-nitrophenoxyhexanoic acid units from p-cyanophenoxyhexanoic acid and p-nitrophenoxyhexanoic acid respectively. Japanese Patent No. 2989175 describes a homopolymer comprised of 3-hydroxy-5-(monofluorophenoxy)valeric acid units or 3-hydroxy-5-(difluorophenoxy)valeric acid units and a copolymer containing at least 3-hydroxy-5-(monofluorophenoxy)pentanoate unit or 3-hydroxy-5-(difluorophenoxy)pentanoate unit and a method for producing such homopolymer or copolymer, reciting that such homopolymer and copolymer can provide water-repellency and stereoregularity with high melting point and good workability.
As an example of unusual-PHA having a cyclohexyl group, Polymers, 30, 1611–1615 (1997) reported that Pseudomonas oleovorans produced such PHA from cyclohexylbutyric acid or cyclohexylvaleric acid.
These mcl-PHA and unusual-PHA are synthesized through an enzymatic polymerization reaction using (R)-3-hydroxyacyl CoA as a substrate. Such 3-hydroxyacyl CoAs are produced through various metabolic pathways (for example, β-oxidation pathway or fatty acid synthesis pathway) in a living organism from different alkanoic acids. The enzyme that catalyzes this polymerization reaction is PHA synthetase (this can be referred to as PHA polymerase or PHA synthase). The following is the reaction route from alkanoic acid to PHA via the β-oxidation pathway and polymerization reaction by PHA synthetase.

On the other hand, when the production is performed through the fatty acid synthesis pathway, it is considered that (R)-3-hydroxyacyl-ACP (ACP means acyl carrier protein) generated in this pathway is converted to (R)-3-hydroxyacyl CoA from which PHA is synthesized by PHA synthetase.
Recently, attempts have been made to synthesize PHA in vitro using PHB synthetase or PHA synthetase isolated from cells.
For example, Proc. Natl. Acad. Sci. USA, 92, 6279–6283 (1995) describes that PHB comprised of 3-hydroxy-n-butyric acid units has been successfully synthesized by using PHB synthetase derived from Alcaligenes eutrophus and 3-hydroxybutyryl CoA as a substrate. In addition, Int. J. Biol. Macromol., 25, 55–60 (1999) describes that PHA comprised of 3-hydroxy-n-butyric acid units or 3-hydroxy-n-valeric acid units has been successfully synthesized by reacting PHB synthetase derived from Alcaligenes eutrophus with 3-hydroxybutyryl CoA or 3-hydroxyvaleryl CoA. Further, this report mentions that, when racemic 3-hydroxybutyryl CoA was reacted with PHB synthetase, PHB comprised of only (R)3-hydroxy-n-butyric acid units was successfully synthesized due to the stereoselectivity of the enzyme. Macromol. Rapid Commun., 21, 77–84 (2000) reported in vitro PHB synthesis using PHB synthetase derived from Alcaligenes eutrophus. 
FEMS Microbiol. Lett., 168, 319–324 (1998) describes that PHB comprised of 3-hydroxy-n-burytic acid units was successfully synthesized by reacting PHB synthetase derived from Chromatium vinosum with 3-hydroxybutyryl CoA.
In Appl. Mircobiol. Biotechnol., 54, 37–43 (2000), PHA comprised of 3-hydroxydecanoic acid is synthesized by reacting PHA synthetase derived from Pseudomonas aeruginosa with 3-hydroxydecanoyl CoA.
As described above, application of biotechnological methods to polymer synthesis may enable synthesis of new polymer compounds which can not be made by conventional organic synthesis or enable imparting new functions and constructs to polymer compounds. In addition, there are many cases where conventional multi-step reaction can be replaced with only one step reaction, so that simplification of the production process, cost reduction, and time saving are expected. Further, this enables consumption reduction of organic solvents, acids, alkalis, surfactants, etc., moderate reaction conditions, and synthesis from non-petroleum raw materials or crude raw materials, thus enables a synthesis process of lower environmental burden and of resource-recycling type. In more detail with synthesis from the crude raw materials, the substrate specificity of the enzyme used as a catalyst in biotechnological synthesis is high in general, so that it is possible to selectively promote a desired reaction even when the low purity raw material is used. Thus, use of waste materials or recycled materials can be expected.
On the other hand, as an elemental technical idea for adding values to polymer compounds, the inventors of the present invention has been focusing on a construct in which a base material is coated with a polymer compound as described above. Coating a certain base material with a polymer compound can provide composite constructs having extremely useful functionality. Conventionally, organic synthesis techniques have been used to produce such constructs, but there are certain limits to such techniques.
If such a construct can be manufactured by using a biotechnological method as described above, it is possible to utilize new polymer compounds that can not be realized by conventional organic synthesis techniques or to impart new functions and constructs to polymer compounds. It is also possible to achieve a making process being less burdening environment and of resource-recycling type at a low cost. For example, utilizing the highly strict molecular recognition ability or stereoselectivity specific to biocatalysts, it is possible to produce capsule constructs or laminated constructs coated with a new functional polymer compound or a polymer compound having extremely high chirality that could not be realized through conventional techniques of the synthetic organic chemistry, through a simple process with low environmental burden.
Therefore, the present invention provides a construct of highly functional polymer compound that can be manufactured by a biotechnological method. The present invention also provides an efficient method for making a construct of a base material coated with a polymer compound usable as a functional composite construct.
Meanwhile, in the field of electrophotography, various methods are known but generally consist of utilizing a photoconductive material, forming an electrical latent image on an image bearing member (photosensitive member) by various means, developing such latent image with toner to obtain a visible image, then transferring if necessary the toner image onto a transfer material such as paper, and fixing the toner image onto the transfer material by heat and/or pressure thereby obtaining a copy. For rendering the electrical latent image visible, there are known a cascade development method, a magnetic brush development method, a pressure development method etc. There is also known a method of employing magnetic toner and a rotary developing sleeve having magnetic poles at the center thereof, thereby causing the magnetic toner to fly from the developing sleeve to the photosensitive member. In the development method for developing the electrostatic latent image, there is known a two-component development method employing two-component developer consisting of toner and carrier, and a one-component development method employing one-component developer consisting solely of toner and not employing carrier.
The colorant generally called toner (hereinafter colorant means a substance which contains a coloring agent as the essential component and may contain other components for providing other functions) contains binder resin and a coloring agent (hereinafter coloring agent means carbon black, pigment, dye or other coloring material itself) as the essential components and further contains magnetic powder or the like if necessary. For a monochromatic copying apparatus there is utilized not only black toner but also monochromatic color toner such as of red, blue, green or brown color, and, in such color toner there is utilized a pigment or a dye for developing respective color instead of the black coloring agent (principally carbon black) in the black toner.
In preparing color toner with such pigment or dye, there is widely employed a method kneading such pigment or dye with binder resin, then executing crushing with a mechanical or air collision crusher and executing classification to achieve desired average particle size and particle size distribution. However, such conventional method has been associated with drawbacks that the coloring agent etc. added in the toner may not be uniformly dispersed depending on the component, or that it may be difficult to produce the toner of a small particle size with a narrow particle size distribution whereby such toner of the conventional method may generate image fog because of the significant loss of charge in certain cases, and that the particle size distribution may become wider to result in selective development thereby leading to deterioration in the image.
Also, since the chargeability and the powder flowability are different for each coloring agent, it is generally conducted to adjust the kind and amount of the internally added charge control agent or the externally added materials, in order to obtain uniform characteristics among the color toners. Also in a full-color copying apparatus, there are employed color toners of magenta, cyan and yellow colors in addition to the black color toner, and, also in this case, there is executed adjustment on the kind and amount of the internally added charge control agent or the externally added material as explained above, in order to obtain uniform characteristics among the color toners. However, such adjustment of the kind and amount of the internally or externally added material as explained in the foregoing for obtaining uniform characteristics in the color toners may practically be quite cumbersome and difficult, so that, both for monochromatic toner and full-color toner, there have been drawbacks of fluctuation in the quality of the copy image or difference in behavior of deterioration among the color toners.
Also in recent years, in the image formation with the full-color electrophotography, though the image quality of the full-color copying apparatus has been improved by the expansion of color presenting area by the improvement in the pigment dispersing technology and by the higher resolution of the image based on the toner of smaller average particle size of 7 to 8 μm, the electrophotographic image is still deficient in the uniformity of image luster, uniformity or image height and reproducibility of halftone image area in comparison with the offset printed image.
In order to resolve the aforementioned unevenness of the image, there is being investigated reduction of the toner amount loaded on the transfer medium based on a further reduction of the diameter of the toner. However, in order to reproduce a density same as that of the printed image with the toner of smaller diameter, it becomes necessary to increase the concentration of the coloring agent to be added in the toner, but an increase in the concentration of the coloring agent in the toner renders it difficult to control the charge amount of the toner, whereby the fluctuation in the charge amount may increase particularly under a high temperature-high humidity or low temperature-low humidity condition or in certain coloring agent, resulting in deterioration of the image quality. Also since the coloring agent enhances the influence on the fusing characteristics of the binder resin, there may result difficulty in the fixing ability such as generation of hot offset phenomenon or decrease of image intensity.
In order to alleviate the aforementioned influence of the coloring agent on the characteristics of toner, the Japanese Patent Application Laid-open Nos. 62-17753, 5-88406, 5-289396 and 5-341574 propose, in toner utilizing resin having surface activating effect as an auxiliary dispersant and having a matrix-domain structure formed by fused phase separation of the resins of different compositions, to disperse the coloring agent only in the domain portion, and the Japanese Patent Application Laid-open No. 63-305367 proposes to cover a nucleus particle, prepared by suspension polymerization and containing the coloring agent in a large amount, with resin of a high electrical resistance not containing the coloring agent by two-step polymerization, thereby controlling the electrical resistance of the toner surface, thus balancing the color developing property and the electrical characteristics.
These proposals can control the dispersion area of the coloring agent in the toner to a certain extent and can therefore relax the influence of the coloring agent on the toner characteristics. However, for achieving phase separation of the resins in the former method, the selection range of the resins is limited because the resin to be used in the matrix and that to be used in the domain have to be mutually unmixable, so that there will result drawbacks that the particle size and distribution of the domain is difficult to control and that it is difficult to include the coloring agent only in the domain in case the concentration of the coloring agent is high or in certain types of the coloring agent.
On the other hand, in the latter method, the toner is prepared by preparing the nucleus particle containing the coloring agent in a large amount by suspension polymerization and then by adding a monomer, but, since the nucleus particle and the monomer constituting the covering resin are of similar types and are mutually well soluble, the coloring agent inevitably migrates to the toner surface to result in deterioration of the toner chargeability and the fixing property. Also in the suspension polymerization, since the concentration of the coloring agent that can be introduced into the nucleus particle is limited, it is difficult to attain a high concentration of the coloring agent in the toner and it is not possible to reduce the size of the nucleus particle. Also in the nucleus particle produced by the suspension polymerization, since the polymerization initiator or the surfactant such as the suspension stabilizer used in a large amount remain in the capsule, the type or amount of the surfactant or the polymerization initiator are limited for certain applications and it may become difficult to attain the desired objective.
Also in the aforementioned producing methods, organic solvent is often used for monomer polymerization or for polymer dissolution, so that it is difficult to use the coloring agent soluble in the organic solvent. Also in case the organic solvent is used in a large amount for mass production, there results a large burden on the facility, human body and environment, such methods are not preferred also in such aspect. Further, these methods are associated with a drawback that the reaction conditions are not easily controllable and that the process steps are also complicated.
On the other hand, bioengineering methods for producing polymer compounds are actively investigated in recent years and are partially employed commercially. As examples of microorganism-origin polymer compounds, there are known polyhydroxyalkanoates (which may hereinafter be abbreviated as PHA) such as poly-3-hydroxy-n-butyric acid (which may hereinafter be abbreviated as PHB) or a copolymer of 3-hydroxy-n-butyric acid and 3-hydroxy-n-valeric acid (which may hereinafter be abbreviated as PHB/V), polysaccharides such as bacteria cellulose or purulan, and polyamino acids such as poly-γ-glutamic acid or polylysin. Such PHA produced by the microorganisms can be utilized for producing various products for example by fusion, like the conventional plastics. It also shows satisfactory matching with the living tissues and is expected in the applications as the soft material for medical use.
It has been reported that various microorganisms produce and accumulate PHA. For example, the production of PHB/V by the microorganisms of Alcaligenes eutropus H16 (ATCC No. 17699), Methylobacterium sp., Paracoccus sp., Alcaligenes sp. and Pseudomonas sp. is reported (Japanese Patent Application Laid-open No. 5-74492, Japanese Patent Publications Nos. 6-15604, 7-14352 and 8-19227 etc.).
It is also disclosed that Comamonas acidovorans (IFO 13852) produce PHA having monomer units including 3-hydroxy-n-butyric acid and 4-hydroxy-n-butyric acid (Japanese Patent Application Laid-open No. 9-191893). It is also disclosed that Aeromonas caviae produce copolymer of 3-hydroxy-n-butyric acid and 3-hydroxyhexanoic acid (Japanese Patent Application Laid-open Nos. 5-93049 and 7-265065).
The biosynthesis of such PHB or PHB/V is executed by a polymerization reaction by an enzyme utilizing, as the substrate, (R)-3-hydroxybutyryl CoA or (R)-3-hydroxyvaleryl CoA produced from various carbon sources through various metabolism paths in the microorganisms. The enzyme catalyzing such polymerization reaction is PHB synthesizing enzyme (also called PHB polymerase or PHB synthase). CoA means coenzyme A having the following chemical structure:

Also in recent years, investigations are actively executed on polyhydroxyalkanoate consisting of 3-hydroxy alkanoic acid unit of a medium-chain-length with 3 to 12 carbon atoms (which may be hereinafter abbreviated as mcl-PHA).
For example, Japanese Patent No. 2642937 discloses that Pseudomonas oleovorans ATCC 29347 supplied with acyclic aliphatic hydrocarbon produces PHA including 3-hydroxy alkanoic acid monomer unit with 6 to 12 carbon atoms. Also, Appl. Environ. Microbiol., 58, 746(1992) reports that Pseudomonas resinovorans produces PHA including 3-hydroxy-n-butyric acid, 3-hydroxy-hexanoic acid, 3-hydroxy-octanoic acid and 3-hydroxy-decanoic acid as monomer units from octanoic acid as the single carbon source, and PHA including 3-hydroxy-n-butyric acid, 3-hydroxy-hexanoic acid, 3-hydroxy-octanoic acid and 3-hydroxy-decanoic acid as monomer units from hexanoic acid as the single carbon source. In these syntheses, the introduction of a 3-hydroxyalkanoic acid unit having a chain length longer than that of the starting fatty acid is assumed to result from a fatty acid synthesis path to be explained later.
Int. J. Biol. Macromol., 16(3), 119(1994) reports that Pseudomonas sp. strain 61-3 produces, from sodium gluconate as a single carbon source, PHA including, as units thereof, 3-hydroxyalkanoic acids such as 3-hydroxy-n-butyric acid, 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid, 3-hydroxy decanoic acid and 3-hydroxydodecanoic acid, and 3-hydroxyalkenoic acids such as 3-hydroxy-5-cis-decenoic acid and 3-hydroxy-5-cis-dodecenoic acid.
The aforementioned PHA is a PHA consisting of a monomer unit having an alkyl radical in the side chain (which may hereinafter be abbreviated as usual-PHA). However, for wider applications, for example for application as functional polymer, PHA having a substituent other than alkyl radical, such as phenyl radical, unsaturated hydrocarbon radical, ester radical, allyl radical, cyano radical, halogenated hydrocarbon, epoxide etc., in the side chain (such PHA may hereinafter be abbreviated as unusual-PHA) is anticipated to be extremely useful.
As an example of biosynthesis of unusual-PHA containing phenyl radical, Macromolecules, 24, 5256–5260(1991), Macromol. Chem., 191, 1957–1965(1990) and Chirality, 3, 492–494(1991) report that Pseudomonas oleovorans produces PHA containing 3-hydroxy-5-phenylvaleric acid as a unit, from 5-phenylvaleric acid. Also Macromolecules, 29, 1762–1766(1996) reports that Pseudomonas oleovorans produces PHA containing 3-hydroxy-5-(4-tolyl)valeric acid as a unit, from 5-(4-tolyl)-valeric acid (i.e. 5-(4-methylphenyl)valeric acid). Also Macromolecules, 32, 2889–2895(1999) reports that Pseudomonas oleovorans produces PHA containing 3-hydroxy-5-(2,4-dinitrophenyl)valeric acid and 3-hydroxy-5-(4-nitrophenyl)valeric acid as units, from 5-(2,4-dinitrophenyl)valeric acid.
Also as examples of unusual-PHA containing phenoxy radical, Macromol. Chem. Phys., 195, 1665–1672(1994) reports that Pseudomonas oleovorans produces PHA containing 3-hydroxy-5-hydroxyvaleric acid and 3-hydroxy-9-phenoxynonanoic acid as the units, from 11-phenoxyundecanoic acid. Also Macromolecules, 29, 3432–3435(1996) reports that Pseudomonas oleovolans produces PHA including 3-hydroxy-4-phenoxybutyric acid unit and 3-hydroxy-6-phenoxyhexanoic acid unit from 6-phenoxyhexanoic acid, also produces PHA including 3-hydroxy-4-phenoxybutyric acid unit, 3-hydroxy-6-phenoxyhexanoic acid unit and 3-hydroxy-8-phenoxyoctanoic acid unit from 8-phenoxyoctanoic acid, and produces PHA including 3-hydroxy-5-phenoxyvaleric acid unit and 3-hydroxy-7-phenoxyheptanoic acid unit from 11-phenoxyundecanoic acid.
Also Can. J. Microbiol., 41, 32–43(1995) reports production of PHA containing 3-hydroxy-6-(p-cyanophenoxy)hexanoic acid or 3-hydroxy-6-(p-nitrophenoxy)hexanoic acid as the monomer unit by Pseudomonas oleovorans ATCC 29347 strain and Pseudomonas putida KT2442 strain, from p-cyanophenoxy hexanoic acid or p-nitrophenoxy hexanoic acid. The Japanese Patent No. 2989175 discloses homopolymer consisting of 3-hydroxy-5-(monofluorophenoxy) valeric acid unit or 3-hydroxy-5-(difluorophenoxy) valeric acid unit, copolymer including at least 3-hydroxy-5-(monofluorophenoxy)pentanoate unit or 3-hydroxy-5-(difluorophenoxy)pentanoate unit and a producing method therefor, and describes the advantages thereof such as providing stereo regularity and water repellency while maintaining high melting point and satisfactory workability.
Also as examples of unusual-PHA having cyclohexyl radical, Macromolecules, 30, 1611–1615(1997) reports that Pseudomonas oleovolans produces such PHA from cyclohexylbutyric acid or cyclohexylvaleric acid.
The biosynthesis of such mcl-PHA or unusual-PHA is executed by a polymerization reaction by an enzyme utilizing, as the substrate, (R)-3-hydroxyacyl CoA produced from various alkanoic acid sources through various metabolism paths (β-oxidation system or fatty acid synthesis path) in the microorganisms. The enzyme catalyzing such polymerization reaction is PHA synthesizing enzyme (also called PHA polymerase or PHA synthase). In the following there are shown reactions from alkanoic acid to PHA through the polymerization reaction by β-oxidation system and PHA synthesizing enzyme.

On the other hand, in case through the fatty acid synthesis path, PHA is assumed to be synthesized similarly by the PHA synthesizing enzyme, from (R)-3-hydroxyacyl CoA converted from (R)-3-hydroxyacyl-ACP (ACP means acyl carrier protein) generated in such path as the substrate.
Recently, it is being tried to take out the aforementioned PHB synthesizing enzyme or PHA synthesizing enzyme from the bacteria and to synthesize PHA in cell-free system (in vitro).
For example, Proc. Natl. Acad. Sci. USA, 92, 6279–6283(1995) reports reacting 3-hydroxybutyryl CoA on PHB synthesizing enzyme derived from Alcaligenes eutrophus to produce PHB consisting of 3-hydroxy-n-butyric acid unit. Also Int. J. Biol. Macromol., 25, 55–60(1999) reports reacting 3-hydroxybutyryl CoA or 3-hydroxyvaleryl CoA on PHB synthesizing enzyme derived from Alcaligenes eutrophus to produce PHA consisting of 3-hydroxy-n-butyric acid unit or 3-hydroxy-n-valeric acid unit. Also this report describes that reaction of racemic 3-hydroxy-butyryl CoA results in synthesis of PHB consisting solely of (R)-3-hydroxy-n-butyric acid unit by the stereo selectivity of the enzyme. Also Macromol. Rapid Commun., 21, 77–84(2000) reports in vitro PHB synthesis utilizing PHB synthesizing enzyme derived from Alcaligenes eutrophus. 
Also FEMS Microbiol. Lett., 168, 319–324(1998) reports reacting 3-hydroxybutyryl CoA on PHB synthesizing enzyme derived from Chromatium vinosum to produce PHB consisting of 3-hydroxy-n-butyric acid unit.
Appl. Microbiol. Biotechnol., 54, 37–43(2000) reports synthesis of PHA consisting of 3-hydroxy-decanoic acid unit by reacting 3-hydroxydecanoyl CoA on PHA synthesizing enzyme of Pseudomonas aeruginosa. 